A temperature-sensitive mutant, K12, isolated from an established Chinese hamster fibroblast cell line, Wg1A, is used to study the coordinated expression of two genes specifically induced by glucose starvation. When K12 cells are incubated at the nonpermissive temperature (40.5~C), the synthesis of several proteins is specifically induced. Two of the proteins (94 and 78 kilodaltons) are identified as glucose-regulated proteins since the same proteins are overproduced in a variety of animal cells when they are starved of glucose. cDNA clones have been constructed with RNA isolated from hamster K12 cells incubated at 40.5~C. Two of the cDNA clones, by criterion of hybrid-select translation, are shown to code for the 94 and 78 kilodalton proteins. Using these cDNA clones as probes, we show that after 4 hrs of incubation at 40.5~C, there is a 10-fold increase in their corresponding mRNA levels. We demonstrate that the kinetics of transcription of these genes directly parallels the accumulation of the mRNA levels, suggesting that the expression of these two genes is coordinatedly and primarily regulated at the transcription level. In addition, the expression of these two genes is stringently regulated by the availability of glucose in the medium and treatment of cells with calcium ionophore A23187. In order to determine the control signals for these genes, a novel eukaryotic hybrid gene has been constructed from the 5' sequence of a rat gene and the bacterial neomycin resistance gene. Following transfection into hamster fibroblasts, the neomycin transcripts can be induced to high levels by the absence of glucose and A23187 treatment. Further, this hybrid gene can also be regulated by temperature when it is introduced into a temperature-sensitive mutant cell line. Our results indicate that the sequence containing the regulatory/promoter function resides within a 1.25-kb region of the rat genomic sequence. (G)